Non-Human Animals Having Humanized FC-Gamma Receptors

ABSTRACT

Genetically modified mice and methods and compositions for making and using the same are provided, wherein the genetic modification comprises humanization of an FcγRI protein.

RELATED APPLICATIONS

This application is a Continuation of application Ser. No. 14/681,617,filed Apr. 8, 2015, which claims the benefit of priority to ProvisionalApplication No. 61/977,037, filed Apr. 8, 2014, each of which is herebyincorporated by reference in its entirety.

BACKGROUND

Fc receptors (FcRs) are proteins found on the surface of cells of theimmune system that carry out a variety of functions of the immune systemin mammals. FcRs exist in a variety of types, on a variety of cells, andmediate a variety of immune functions such as, for example, binding toantibodies that are attached to infected cells or invading pathogens,stimulating phagocytic or cytotoxic cells to destroy microbes, orinfected cells by antibody-mediated phagocytosis or antibody-dependentcell-mediated cytotoxicity (ADCC).

ADCC is a process whereby effector cells of the immune system lyse atarget cell bound by antibodies. This process depends on prior exposureto a foreign antigen or cell, resulting in an antibody response. ADCCcan be mediated through effector cells such as, for example, naturalkiller (NK) cells, by binding of FcR expressed on the surface of theeffector cell to the Fc portion of the antibody which itself is bound tothe foreign antigen or cell. High affinity FcγR1 receptor signalingplays an important role in immune system regulation and effector cellfunction.

SUMMARY OF INVENTION

The present invention encompasses the recognition that it is desirableto engineer non-human animals, such as mice, to express a human orhybrid FcγRI protein to permit experimentation on human immune effectorresponses that could not be performed in humans.

The present invention also encompasses the recognition that it isdesirable to replace the endogenous mouse FcγRI gene with a humanized(human or hybrid) FcγRI gene.

In some embodiments, the invention provides a mouse that expresses aFcγRI protein comprising the extracellular portion of a human FcγRIαchain and the intracellular portion of a mouse FcγRIα chain. In someembodiments, an extracellular portion of a human FcγRIα chain comprisesan EC1 domain, EC2 domain, EC3 domain, or combinations thereof.

In some embodiments, the EC1 domain is encoded by an exon at least 50%,70%, 85%, 90% or 95% identical to exon 3 of SEQ ID NO: 3.

In some embodiments, the EC2 domain is encoded by an exon at least 50%,70%, 85%, 90%, or 95% identical to exon 4 of SEQ ID NO: 3

In some embodiments, the EC3 domain is encoded by an exon at least 50%,70%, 85%, 90%, or 95% identical to exon 5 of SEQ ID NO: 3.

In some embodiments, the invention provides a mouse that expresses aFcγRI protein comprising the extracellular portion of a human FcγRIαchain and the intracellular portion of a mouse FcγRIα chain wherein themouse does not detectably express a full-length mouse FcγRIα chain. Insome embodiments, the intracellular portion of a FcγRIα chain comprisesa cytoplasmic domain of a mouse FcγRIα chain in whole or in part. Insome embodiments, a mouse further expresses a FcγRIα chain comprising amouse FcγRIα chain transmembrane domain in whole or in part.

In some embodiments, the invention provides a mouse that expresses anFcγRIα chain amino acid sequence at least 70%, 85%, 90%, or 95%identical to SEQ ID NO: 5. In some embodiments, human or hybrid FcγRIprotein is detectably expressed on monocytes, macrophages, neutrophils,dendritic cells, and/or combinations thereof.

In some embodiments, human FcγRI protein level is increased byadministration of murine granulocyte colony stimulating factor (mG-CSF).In some embodiments, mouse FcγRI protein level is not increased inmonocytes, neutrophils, or dendritic cells by administration of murinegranulocyte colony stimulating factor (mG-CSF).

In some embodiments, the invention provides a mouse that expresses anFcγRI gene that comprises one or more exons of a human FcγRI geneoperably linked to one or more exons of a mouse FcγRI gene. In someembodiments, exons of a human FcγRI gene encode one or moreextracellular portions of human FcγRI protein. In some embodiments,exons of a mouse FcγRI gene encode one or more intracellular portions ofa mouse FcγRI protein. In some embodiments, exons of a human FcγRI areselected from the group consisting of exons 3, 4, 5, and combinationsthereof.

In some embodiments, an intracellular portion of a mouse FcγRI isoperably linked to one or more mouse intracellular signaling cascades.

In some embodiments, the invention provides a mouse that expresses ahuman FcγRI, wherein germ line cells of the mouse lack a functionalmouse FcγRI gene. In some embodiment the invention provides a mouse thatexpresses a human FcγRI, wherein germ line cells of the mouse lack anymouse FcγRI gene.

In some embodiments, the invention provides an embryonic stem cell whosegenome comprises a FcγRI gene that encodes the extracellular portion ofa human FcγRI protein and the intracellular portion of a mouse FcγRIprotein. In some embodiments, an FcγRI gene comprises exons 3, 4, and 5of a human FcγRI gene. In some embodiments, an FcγRI gene furthercomprises one or more human 5′ untranslated regions flanking humanexon 1. In some embodiments, an extracellular portion of a human FcγRIprotein comprises one or more of EC1, EC2, and EC3.

In some embodiments, the invention provides an embryonic stem cell whosegenome comprises an FcγRIα chain amino acid sequence at least 70%, 85%,90%, or 95% identical to SEQ ID NO: 5.

In some embodiments, an embryonic stem cell comprises an FcγRI genecomprising amino acid residues of exon 6 of a mouse FcγRI gene. In someembodiments, an intracellular portion of a mouse FcγRI protein comprisesthe cytoplasmic domain of a mouse FcγRI protein in whole or in part.

In some embodiments, the invention provides an embryonic stem cellcomprising a human FcγRI gene wherein the human FcγRI gene is positionedat an endogenous FcγRI locus that appears in a mouse genome as found innature.

In some embodiments, the invention provides a mouse embryo generatedfrom an embryonic stem as described herein.

In some embodiments, the invention provides for use of a mouse embryonicstem cell as described herein to make a transgenic mouse.

In some embodiments, methods of making a mouse that expresses FcγRIprotein comprising an extracellular portion of a human FcγRI protein andan intracellular portion of a mouse FcγRI protein are disclosed, themethod comprising steps of: (a) obtaining a mouse embryonic stem cell;(b) replacing in the embryonic cell an endogenous mouse FcγRI gene witha genomic fragment comprising a nucleic acid molecule that encodes aportion of human FcγRI protein having human extracellular regions; and(c) creating the mouse using the embryonic cell of (b).

In some embodiments, the invention provides a genomic fragmentcomprising a nucleic acid molecule that encodes a portion of human FcγRIprotein having human extracellular regions and a nucleic acid moleculethat encodes an intracellular portion of a mouse FcγRI protein.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

Other features, objects, and advantages of the present invention areapparent in the detailed description that follows. It should beunderstood, however, that the detailed description, while indicatingembodiments of the present invention, is given by way of illustrationonly, not limitation. Various changes and modifications within the scopeof the invention will become apparent to those skilled in the art fromthe detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings included herein are for illustration purposes only not forlimitation.

FIG. 1 shows genotyping of the FcγRI mouse allele in experimental miceand wild-type controls.

FIG. 2 shows relative copy number of the human FcγRI gene inexperimental mice and wild-type controls.

FIG. 3 shows transformed data for gene copy numbers based on Het (±)mice having one copy of the human FcγRI and calibrated by average ΔCt.

FIG. 4 shows expression of FcγRI receptors in cells from human blooddonors.

FIG. 5 shows a schematic illustration (not to scale) of a human FcγRIgene, a mouse FcγRI gene, and a humanized FcγRI gene.

FIG. 6 shows a schematic illustration (not to scale) of a process ofpreparing a MAID 6074 cassette.

FIG. 7 shows mice having a humanized FcγRI gene exhibit normal cellfrequencies in spleen and blood.

FIG. 8 shows myeloid spleen populations in mice having a humanized FcγRIgene.

FIG. 9 shows a loss of murine FcγRI expression on macrophages from thespleen of mice having a humanized FcγRI gene.

FIG. 10 shows a gain of human FcγRI expression on monocytes from thespleen of mice having a humanized FcγRI gene.

FIG. 11 shows the gating strategy during FACS analysis of macrophagespurified from the peritoneal cavity of mice having endogenous mouseFcγRI genes (MAID 6074 WT) and mice homozygous for a humanized FcγRIgene (MAID 6074 HO).

FIG. 12 shows expression of human FcγRI and mouse FcγRI in macrophagesfrom the peritoneal cavity of mice having endogenous mouse FcγRI genes(MAID 6074 WT) and mice homozygous for a humanized FcγRI gene (MAID 6074HO).

FIG. 13 shows the gating strategy during FACS analysis of bone marrowderived macrophages of mice having endogenous mouse FcγRI genes (MAID6074 WT) and mice homozygous for a humanized FcγRI gene (MAID 6074 HO).

FIG. 14 shows expression of human FcγRI and mouse FcγRI in bonemarrow-derived macrophages of mice having endogenous mouse FcγRI genes(MAID 6074 WT) and mice homozygous for a humanized FcγRI gene (MAID 6074HO).

FIG. 15 shows the gating strategy during FACS analysis of bone marrowderived macrophages of mice having endogenous mouse FcγRI genes (Control75/25) and mice heterozygous for a humanized FcγRI gene (MAID 6074 HET).

FIG. 16 shows expression of human FcγRI and mouse FcγRI in bonemarrow-derived macrophages of mice having endogenous mouse FcγRI genes(Control 75/25) and mice heterozygous for a humanized FcγRI gene (MAID6074 HET).

FIG. 17 shows myeloid blood populations in MAID 6074 HO mice compared toMAID 6074 WT 48 hours after treatment with PBS.

FIG. 18 shows myeloid blood populations in MAID 6074 HO mice compared toMAID 6074 WT mice 48 hours after treatment with mG-CSF.

FIG. 19 shows a lack of human FcγRI expression in the blood of MAID 6074WT mice and MAID 6074 HO mice 48 hours after treatment with PBS.

FIG. 20 shows human FcγRI expression in the blood of MAID 6074 HO micecompared to MAID 6074 WT mice 48 hours after treatment with mG-CSF.

FIG. 21 shows lack of murine FcγRI expression in the blood of MAID 6074WT and MAID 6074 HO mice 48 hours after treatment with PBS.

FIG. 22 shows murine FcγRI expression in the blood of MAID 6074 WTcompared to MAID 6074 HO mice 48 hours after treatment with mG-CSF.

FIG. 23 shows myeloid splenic populations in MAID 6074 HO compared toMAID 6074 WT mice 48 hours after treatment with PBS.

FIG. 24 shows myeloid splenic populations in MAID 6074 HO mice comparedto MAID 6074 WT mice 48 hours after treatment with mG-CSF.

FIG. 25 shows a lack of human FcγRI expression in splenic monocytes inMAID 6074 HO mice and 6074 WT mice 48 hours after treatment with PBS.

FIG. 26 shows human FcγRI expression in the spleen of MAID 6074 HO micecompared to MAID 6074 WT mice 48 hours after treatment with mG-CSF.

FIG. 27 shows murine FcγRI expression in the spleen of MAID 6074 WT micecompared to MAID 6074 HO mice 48 hours after treatment with PBS.

FIG. 28 shows murine FcγRI expression in the spleen of MAID 6074 WT micecompared to MAID 6074 HO mice 48 hours after treatment with mG-CSF.

FIG. 29 shows a summary of human FcγRI expression in cell populations ofMAID 6074 WT and MAID 6074 HO mice after treatment with PBS or mG-CSF.

FIG. 30 shows upregulation of human FcγRI mRNA induced by mG-CSF in MAID6074 HO mouse blood and spleen normalized to mHPRT1.

FIG. 31 shows upregulation of human FcγRI mRNA induced by mG-CSF in MAID6074 HO mouse blood and spleen.

FIG. 32 depicts a schematic for and exemplary strategy for thehumanization of mouse FcγRI.

DEFINITIONS

This invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention is defined bythe claims.

Unless defined otherwise, all terms and phrases used herein include themeanings that the terms and phrases have attained in the art, unless thecontrary is clearly indicated or clearly apparent from the context inwhich the term or phrase is used. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, particular methods andmaterials are now described.

The term “approximately” as applied herein to one or more values ofinterest, refers to a value that is similar to a stated reference value.In certain embodiments, the term “approximately” or “about” refers to arange of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less ineither direction (greater than or less than) of the stated referencevalue unless otherwise stated or otherwise evident from the context(except where such number would exceed 100% of a possible value).

The term “biologically active” as used herein refers to a characteristicof any agent that has activity in a biological system, in vitro or invivo (e.g., in an organism). For instance, an agent that, when presentin an organism, has a biological effect within that organism, isconsidered to be biologically active. In particular embodiments, where aprotein or polypeptide is biologically active, a portion of that proteinor polypeptide that shares at least one biological activity of theprotein or polypeptide is typically referred to as a “biologicallyactive” portion.

The term “comparable” as used herein refers to two or more agents,entities, situations, sets of conditions, etc. that may not be identicalto one another but that are sufficiently similar to permit comparisonthere between so that conclusions may reasonably be drawn based ondifferences or similarities observed. Those of ordinary skill in the artwill understand, in context, what degree of identity is required in anygiven circumstance for two or more such agents, entities, situations,sets of conditions, etc. to be considered comparable.

The term “conservative” is used herein to describe a conservative aminoacid substitution refers to substitution of an amino acid residue byanother amino acid residue having a side chain R group with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of interest of a protein, for example, the abilityof a receptor to bind to a ligand. Examples of groups of amino acidsthat have side chains with similar chemical properties include aliphaticside chains such as glycine, alanine, valine, leucine, and isoleucine;aliphatic-hydroxyl side chains such as serine and threonine;amide-containing side chains such as asparagine and glutamine; aromaticside chains such as phenylalanine, tyrosine, and tryptophan; basic sidechains such as lysine, arginine, and histidine; acidic side chains suchas aspartic acid and glutamic acid; and, sulfur-containing side chainssuch as cysteine and methionine. Conservative amino acids substitutiongroups include, for example, valine/leucine/isoleucine,phenylalanine/tyrosine, lysine/arginine, alanine/valine,glutamate/aspartate, and asparagine/glutamine. In some embodiments, aconservative amino acid substitution can be substitution of any nativeresidue in a protein with alanine, as used in, for example, alaninescanning mutagenesis. In some embodiments, a conservative substitutionis made that has a positive value in the PAM250 log-likelihood matrixdisclosed in Gonnet et al. (1992) Exhaustive Matching of the EntireProtein Sequence Database, Science 256:1443-45, hereby incorporated byreference. In some embodiments, the substitution is a moderatelyconservative substitution wherein the substitution has a nonnegativevalue in the PAM250 log-likelihood matrix.

The term “disruption” as used herein refers to the result of ahomologous recombination event with a DNA molecule (e.g., with anendogenous homologous sequence such as a gene or gene locus. In someembodiments, a disruption may achieve or represent an insertion,deletion, substitution, replacement, missense mutation, or a frame-shiftof a DNA sequence(s), or any combination thereof. Insertions may includethe insertion of entire genes or fragments of genes, e.g. exons, whichmay be of an origin other than the endogenous sequence. In someembodiments, a disruption may increase expression and/or activity of agene or gene product (e.g., of a protein encoded by a gene). In someembodiments, a disruption may decrease expression and/or activity of agene or gene product. In some embodiments, a disruption may altersequence of a gene or an encoded gene product (e.g., an encodedprotein). In some embodiments, a disruption may truncate or fragment agene or an encoded gene product (e.g., an encoded protein). In someembodiments, a disruption may extend a gene or an encoded gene product;in some such embodiments, a disruption may achieve assembly of a fusionprotein. In some embodiments, a disruption may affect level but notactivity of a gene or gene product. In some embodiments, a disruptionmay affect activity but not level of a gene or gene product. In someembodiments, a disruption may have no significant effect on level of agene or gene product. In some embodiments, a disruption may have nosignificant effect on activity of a gene or gene product. In someembodiments, a disruption may have no significant effect on either levelor activity of a gene or gene product.

The phrase “endogenous locus” or “endogenous gene” as used herein refersto a genetic locus found in a parent or reference organism prior tointroduction of a disruption, deletion, replacement, alteration, ormodification as described herein. In some embodiments, the endogenouslocus has a sequence found in nature. In some embodiments, theendogenous locus is wild type. In some embodiments, the referenceorganism is a wild-type organism. In some embodiments, the referenceorganism is an engineered organism. In some embodiments, the referenceorganism is a laboratory-bred organism (whether wild-type orengineered).

The phrase “endogenous promoter” as used herein refers to a promoterthat is naturally associated, e.g., in a wild-type organism, with anendogenous gene.

The term “FcγRI protein” as used herein refers to a high affinityimmunoglobulin Fc receptor comprising an α chain having threeextracellular domains, a transmembrane domain, and an intracellulardomain.

By way of illustration, representative nucleotide and amino acidsequences of a mouse and human FcγRIα genes are provided in Table 3.Persons of skill upon reading this disclosure will recognize that one ormore endogenous FcγRI receptor genes in a genome (or all) can bereplaced by one or more heterologous FcγRI genes (e.g., polymorphicvariants, subtypes or mutants, genes from another species, etc.).

A “FcγRI-expressing cell” as used herein refers to a cell that expressesFcγRT. In some embodiments, an FcγRI-expressing cell expresses a FcγRIreceptor on its surface. In some embodiments, a FcγRI receptor isexpressed on the surface of the cell in an amount sufficient to mediatecell-to-cell interactions via the FcγRI protein expressed on the surfaceof the cell. Exemplary FcγRI-expressing cells include, lymphocytes,myeloid cells, macrophages, neutrophils, and natural killer (NK) cells.FcγRI-expressing cells regulate the interaction of immune cells toregulate the immune response to various foreign antigens or pathogens.In some embodiments, non-human animals of the present inventiondemonstrate immune cell regulation via humanized FcγRI receptorsexpressed on the surface of one more cells of the non-human animal.

The term “heterologous” as used herein refers to an agent or entity froma different source. For example, when used in reference to apolypeptide, gene, or gene product or present in a particular cell ororganism, the term clarifies that the relevant polypeptide, gene, orgene product 1) was engineered by the hand of man; 2) was introducedinto the cell or organism (or a precursor thereof) through the hand ofman (e.g., via genetic engineering); and/or 3) is not naturally producedby or present in the relevant cell or organism (e.g., the relevant celltype or organism type).

The term “host cell” as used herein refers to a cell into which aheterologous (e.g., exogenous) nucleic acid or protein has beenintroduced. Persons of skill upon reading this disclosure willunderstand that such terms refer not only to the particular subjectcell, but also is used to refer to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein. In some embodiments, a host cell isor comprises a prokaryotic or eukaryotic cell. In general, a host cellis any cell that is suitable for receiving and/or producing aheterologous nucleic acid or protein, regardless of the Kingdom of lifeto which the cell is designated. Exemplary cells include those ofprokaryotes and eukaryotes (single-cell or multiple-cell), bacterialcells (e.g., strains ofE. coli, Bacillus spp., Streptomyces spp., etc.),mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S.pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells(e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni,etc.), non-human animal cells, human cells, or cell fusions such as, forexample, hybridomas or quadromas. In some embodiments, the cell is ahuman, monkey, ape, hamster, rat, or mouse cell. In some embodiments,the cell is eukaryotic and is selected from the following cells: CHO(e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell,Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK),HeLa, HepG2, WI38, MRC 5, Co1o205, HB 8065, HL-60, (e.g., BHK21),Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell,SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myelomacell, tumor cell, and a cell line derived from an aforementioned cell.In some embodiments, the cell comprises one or more viral genes, e.g., aretinal cell that expresses a viral gene (e.g., a PER.C6™ cell). In someembodiments, a host cell is or comprises an isolated cell. In someembodiments, a host cell is part of a tissue. In some embodiments, ahost cell is part of an organism.

The phrase “human FcγRI gene” as used herein refers to a nucleotidesequence encoding a fully human, substantially human, or humanizedportion of an FcγRI protein depending on context. In some embodiments, a“human FcγRI” gene refers to a humanized FcγRI gene as contrasted with afully mouse FcγRI gene. In some embodiments, a human FcγRI gene containsone or more substitutions, additions, deletions, or mutations. In someembodiments a human FcγRI gene comprises FcγRIA (CD64A), FcγRIB (CD64B),FcγRIC (CD64C), or combinations thereof

The phrase “human FcγRI protein” refers to a protein encoded by a fullyhuman, substantially human, or humanized FcγRI gene depending oncontext. In some embodiments, a “human FcγRI” protein refers to ahumanized FcγRI protein as contrasted with a fully mouse FcγRI protein.In some embodiments, a human FcγRI protein comprises one or more aminoacid substitutions, additions, deletions, or mutations. In someembodiments, a FcγRI protein comprises FcγRIA (CD64A), FcγRIB (CD64B),FcγRIC (CD64C), or combinations thereof

The phrase “hybrid FcγRI gene” or “hybrid FcγRI protein” refers to aFcγRI gene or protein that includes an FcγRI sequence of at least twodifferent species of animals. In some embodiments, a hybrid FcγRI geneincludes a portion of a human nucleic acid sequence and a portion of amouse nucleic acid sequence. In some embodiments, a hybrid FcγRI proteinincludes a portion of human amino acid sequence and a portion of a mouseamino acid sequence.

The term “humanized”, is used herein in accordance with itsart-understood meaning to refer to nucleic acids or proteins whosestructures (i.e., nucleotide or amino acid sequences) include portionsthat correspond substantially or identically with structures of aparticular gene or protein found in nature in a non-human animal, andalso include portions that differ from that found in the relevantparticular non-human gene or protein and instead correspond more closelywith comparable structures found in a corresponding human gene orprotein. In some embodiments, a “humanized” gene is one that encodes apolypeptide having substantially the amino acid sequence as that of ahuman polypeptide (e.g., a human protein or portion thereof—e.g.,characteristic portion thereof). To give but one example, in the case ofa membrane receptor, a “humanized” gene may encode a polypeptide havingan extracellular portion having an amino acid sequence as that of ahuman extracellular portion and the remaining sequence as that of anon-human (e.g., mouse) polypeptide. In some embodiments, a humanizedgene comprises at least a portion of an DNA sequence of a human gene. Insome embodiment, a humanized gene comprises an entire DNA sequence of ahuman gene. In some embodiments, a humanized protein comprises asequence having a portion that appears in a human protein. In someembodiments, a humanized protein comprises an entire sequence of a humanprotein and is expressed from an endogenous locus of a non-human animalthat corresponds to the homolog or ortholog of the human gene.

The term “identity” as used herein in connection with a comparison ofsequences, refers to identity as determined by a number of differentalgorithms known in the art that can be used to measure nucleotideand/or amino acid sequence identity. In some embodiments, identities asdescribed herein are determined using a ClustalW v. 1.83 (slow)alignment employing an open gap penalty of 10.0, an extend gap penaltyof 0.1, and using a Gonnet similarity matrix (MACVECTOR™ 10.0.2,MacVector Inc., 2008).

The terms “intracellular signal cascade” or “intracellular signaltransduction” as used herein refers to a transmission of signal from acell surface to one or more intracellular targets. In some embodiments,intracellular signal transduction comprises a physiological response ina cell that is elicited by the binding of a target molecule (e.g., animmunoglobulin Fc region) to an extracellular component of a FcγR1receptor.

The term “isolated” as used herein refers to a substance and/or entitythat has been (1) separated from at least some of the components withwhich it was associated when initially produced (whether in natureand/or in an experimental setting), and/or (2) designed, produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or more than about 99% of the other componentswith which they were initially associated. In some embodiments, isolatedagents are about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 9′7%, about 98%, about 99%,or more than about 99% pure. As used herein, a substance is “pure” if itis substantially free of other components. In some embodiments, as willbe understood by those skilled in the art, a substance may still beconsidered “isolated” or even “pure”, after having been combined withcertain other components such as, for example, one or more carriers orexcipients (e.g., buffer, solvent, water, etc.); in such embodiments,percent isolation or purity of the substance is calculated withoutincluding such carriers or excipients. To give but one example, in someembodiments, a biological polymer such as a polypeptide orpolynucleotide that occurs in nature is considered to be “isolated”when, a) by virtue of its origin or source of derivation is notassociated with some or all of the components that accompany it in itsnative state in nature; b) it is substantially free of otherpolypeptides or nucleic acids of the same species from the species thatproduces it in nature; c) is expressed by or is otherwise in associationwith components from a cell or other expression system that is not ofthe species that produces it in nature. Thus, for instance, in someembodiments, a polypeptide that is chemically synthesized or issynthesized in a cellular system different from that which produces itin nature is considered to be an “isolated” polypeptide. Alternativelyor additionally, in some embodiments, a polypeptide that has beensubjected to one or more purification techniques may be considered to bean “isolated” polypeptide to the extent that it has been separated fromother components a) with which it is associated in nature; and/or b)with which it was associated when initially produced.

The phrase “mouse FcγRI gene” as used herein refers to a gene comprisinga nucleic molecule as shown in SEQ ID NO: 1, or a nucleic acid moleculehaving substantial identity to a molecule as shown in SEQ ID NO: 1.

The phrase “mouse FcγRI protein” as used herein refers to a proteincomprising an amino acid sequence as shown in SEQ ID NO: 2, including aprotein having substantial identity to a protein as shown in SEQ ID NO:2.

The phrase “non-human animal” as used herein refers to any vertebrateorganism that is not a human. In some embodiments, a non-human animal isa cyclostome, a bony fish, a cartilaginous fish (e.g., a shark or aray), an amphibian, a reptile, a mammal, and a bird. In someembodiments, a non-human mammal is a primate, a goat, a sheep, a pig, adog, a cow, or a rodent. In some embodiments, a non-human animal is arodent such as a rat or a mouse.

The phrase “nucleic acid” as used herein in its broadest sense, refersto any compound and/or substance that is or can be incorporated into anoligonucleotide chain. In some embodiments, a nucleic acid is a compoundand/or substance that is or can be incorporated into an oligonucleotidechain via a phosphodiester linkage. As will be clear from context, insome embodiments, “nucleic acid” refers to individual nucleic acidresidues (e.g., nucleotides and/or nucleosides); in some embodiments,“nucleic acid” refers to an oligonucleotide chain comprising individualnucleic acid residues. In some embodiments, a “nucleic acid” is orcomprises RNA; in some embodiments, a “nucleic acid” is or comprisesDNA. In some embodiments, a nucleic acid is, comprises, or consists ofone or more natural nucleic acid residues. In some embodiments, anucleic acid is, comprises, or consists of one or more nucleic acidanalogs. In some embodiments, a nucleic acid analog differs from anucleic acid in that it does not utilize a phosphodiester backbone. Forexample, in some embodiments, a nucleic acid is, comprises, or consistsof one or more “peptide nucleic acids”, which are known in the art andhave peptide bonds instead of phosphodiester bonds in the backbone, areconsidered within the scope of the present invention. Alternatively oradditionally, in some embodiments, a nucleic acid has one or morephosphorothioate and/or 5′-N-phosphoramidite linkages rather thanphosphodiester bonds. In some embodiments, a nucleic acid is, comprises,or consists of one or more natural nucleosides (e.g., adenosine,thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine,deoxyguanosine, and deoxycytidine). In some embodiments, a nucleic acidis, comprises, or consists of one or more nucleoside analogs (e.g.,2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine,C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine,C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine,8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, 2-thiocytidine,methylated bases, intercalated bases, and combinations thereof). In someembodiments, a nucleic acid comprises one or more modified sugars (e.g.,2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose) ascompared with those in natural nucleic acids. In some embodiments, anucleic acid has a nucleotide sequence that encodes a functional geneproduct such as an RNA or protein. In some embodiments, a nucleic acidincludes one or more introns. In some embodiments, nucleic acids areprepared by one or more of isolation from a natural source, enzymaticsynthesis by polymerization based on a complementary template (in vivoor in vitro), reproduction in a recombinant cell or system, and chemicalsynthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900,1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residueslong. In some embodiments, a nucleic acid is single stranded; in someembodiments, a nucleic acid is double stranded. In some embodiments anucleic acid has a nucleotide sequence comprising at least one elementthat encodes, or is the complement of a sequence that encodes, apolypeptide. In some embodiments, a nucleic acid has enzymatic activity.

The phrase “operably linked” as used herein refers to a juxtapositionwherein the components described are in a relationship permitting themto function in their intended manner. A control sequence “operablylinked” to a coding sequence is ligated in such a way that expression ofthe coding sequence is achieved under conditions compatible with thecontrol sequences. “Operably linked” sequences include both expressioncontrol sequences that are contiguous with the gene of interest andexpression control sequences that act in trans or at a distance tocontrol the gene of interest. The term “expression control sequence” asused herein refers to polynucleotide sequences which are necessary toeffect the expression and processing of coding sequences to which theyare ligated. Expression control sequences include appropriatetranscription initiation, termination, promoter and enhancer sequences;efficient RNA processing signals such as splicing and polyadenylationsignals; sequences that stabilize cytoplasmic mRNA; sequences thatenhance translation efficiency (i.e., Kozak consensus sequence);sequences that enhance protein stability; and when desired, sequencesthat enhance protein secretion. The nature of such control sequencesdiffers depending upon the host organism. For example, in prokaryotes,such control sequences generally include promoter, ribosomal bindingsite, and transcription termination sequence, while in eukaryotes,typically, such control sequences include promoters and transcriptiontermination sequence. The term “control sequences” is intended toinclude components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

The term “polypeptide” as used herein refers to any polymeric chain ofamino acids. In some embodiments, a polypeptide has an amino acidsequence that occurs in nature. In some embodiments, a polypeptide hasan amino acid sequence that does not occur in nature. In someembodiments, a polypeptide has an amino acid sequence that is engineeredin that it is designed and/or produced through action of the hand ofman.

The term “recombinant” as used herein is intended to refer topolypeptides (e.g., signal-regulatory proteins as described herein) thatare designed, engineered, prepared, expressed, created or isolated byrecombinant means, such as polypeptides expressed using a recombinantexpression vector transfected into a host cell, polypeptides isolatedfrom a recombinant, combinatorial human polypeptide library (HoogenboomH. R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E., (2002)Clin. Biochem. 35:425-445; Gavilondo J. V., and Larrick J. W. (2002)BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) ImmunologyToday 21:371-378), antibodies isolated from an animal (e.g., a mouse)that is transgenic for human immunoglobulin genes (see e.g., Taylor, L.D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., andGreen L. L. (2002) Current Opinion in Biotechnology 13:593-597; LittleM. et al (2000) Immunology Today 21:364-370) or polypeptides prepared,expressed, created or isolated by any other means that involves splicingselected sequence elements to one another. In some embodiments, one ormore of such selected sequence elements is found in nature. In someembodiments, one or more of such selected sequence elements is designedin silico. In some embodiments, one or more such selected sequenceelements results from mutagenesis (e.g., in vivo or in vitro) of a knownsequence element, e.g., from a natural or synthetic source. For example,in some embodiments, a recombinant polypeptide is comprised of sequencesfound in the genome of a source organism of interest (e.g., human,mouse, etc.). In some embodiments, a recombinant polypeptide has anamino acid sequence that resulted from mutagenesis (e.g., in vitro or invivo, for example in a non-human animal), so that the amino acidsequences of the recombinant polypeptides are sequences that, whileoriginating from and related to polypeptides sequences, may notnaturally exist within the genome of a non-human animal in vivo.

The term “replacement” is used herein to refer to a process throughwhich a “replaced” nucleic acid sequence (e.g., a gene) found in a hostlocus (e.g., in a genome) is removed from that locus and a different,“replacement” nucleic acid is located in its place. In some embodiments,the replaced nucleic acid sequence and the replacement nucleic acidsequences are comparable to one another in that, for example, they arehomologous to one another and/or contain corresponding elements (e.g.,protein-coding elements, regulatory elements, etc.). In someembodiments, a replaced nucleic acid sequence includes one or more of apromoter, an enhancer, a splice donor site, a splice receiver site, anintron, an exon, an untranslated region (UTR); in some embodiments, areplacement nucleic acid sequence includes one or more coding sequences.In some embodiments, a replacement nucleic acid sequence is a homolog ofthe replaced nucleic acid sequence. In some embodiments, a replacementnucleic acid sequence is an ortholog of the replaced sequence. In someembodiments, a replacement nucleic acid sequence is or comprises a humannucleic acid sequence. In some embodiments, including where thereplacement nucleic acid sequence is or comprises a human nucleic acidsequence, the replaced nucleic acid sequence is or comprises a rodentsequence (e.g., a mouse sequence). The nucleic acid sequence so placedmay include one or more regulatory sequences that are part of sourcenucleic acid sequence used to obtain the sequence so placed (e.g.,promoters, enhancers, 5′- or 3′-untranslated regions, etc.). Forexample, in various embodiments, the replacement is a substitution of anendogenous sequence with a heterologous sequence that results in theproduction of a gene product from the nucleic acid sequence so placed(comprising the heterologous sequence), but not expression of theendogenous sequence; the replacement is of an endogenous genomicsequence with a nucleic acid sequence that encodes a protein that has asimilar function as a protein encoded by the endogenous sequence (e.g.,the endogenous genomic sequence encodes a FcγRI protein, and the DNAfragment encodes one or more human FcγRI proteins). In variousembodiments, an endogenous gene or fragment thereof is replaced with acorresponding human gene or fragment thereof. A corresponding human geneor fragment thereof is a human gene or fragment that is an ortholog of,or is substantially similar or the same in structure and/or function, asthe endogenous gene or fragment thereof that is replaced.

The term “substantially” as used herein refers to the qualitativecondition of exhibiting total or near-total extent or degree of acharacteristic or property of interest. One of ordinary skill in thebiological arts will understand that biological and chemical phenomenararely, if ever, go to completion and/or proceed to completeness orachieve or avoid an absolute result. The term “substantially” istherefore used herein to capture the potential lack of completenessinherent in many biological and chemical phenomena.

The phrase “substantial homology” as used herein refers to a comparisonbetween amino acid or nucleic acid sequences. As will be appreciated bythose of ordinary skill in the art, two sequences are generallyconsidered to be “substantially homologous” if they contain homologousresidues in corresponding positions. Homologous residues may beidentical residues. Alternatively, homologous residues may benon-identical residues will appropriately similar structural and/orfunctional characteristics. For example, as is well known by those ofordinary skill in the art, certain amino acids are typically classifiedas “hydrophobic” or “hydrophilic” amino acids, and/or as having “polar”or “non-polar” side chains. Substitution of one amino acid for anotherof the same type may often be considered a “homologous” substitution.Typical amino acid categorizations are summarized in Table 1 and 2.

TABLE 1 Alanine Ala A nonpolar neutral 1.8 Arginine Arg R polar positive−4.5 Asparagine Asn N polar neutral −3.5 Aspartic acid Asp D polarnegative −3.5 Cysteine Cys C nonpolar neutral 2.5 Glutamic acid Glu Epolar negative −3.5 Glutamine Gln Q polar neutral −3.5 Glycine Gly Gnonpolar neutral −0.4 Histidine His H polar positive −3.2 Isoleucine IleI nonpolar neutral 4.5 Leucine Leu L nonpolar neutral 3.8 Lysine Lys Kpolar positive −3.9 Methionine Met M nonpolar neutral 1.9 PhenylalaninePhe F nonpolar neutral 2.8 Proline Pro P nonpolar neutral −1.6 SerineSer S polar neutral −0.8 Threonine Thr T polar neutral −0.7 TryptophanTrp W nonpolar neutral −0.9 Tyrosine Tyr Y polar neutral −1.3 Valine ValV nonpolar neutral 4.2

TABLE 2 Ambiguous Amino Acids 3-Letter 1-Letter Asparagine or asparticacid Asx B Glutamine or glutamic acid Glx Z Leucine or Isoleucine Xle JUnspecified or unknown amino acid Xaa X

As is well known in this art, amino acid or nucleic acid sequences maybe compared using any of a variety of algorithms, including thoseavailable in commercial computer programs such as BLASTN for nucleotidesequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acidsequences. Exemplary such programs are described in Altschul, et al.,Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990;Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLASTand PSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al.,Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins,Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods andProtocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999.In addition to identifying homologous sequences, the programs mentionedabove typically provide an indication of the degree of homology. In someembodiments, two sequences are considered to be substantially homologousif at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues arehomologous over a relevant stretch of residues. In some embodiments, therelevant stretch is a complete sequence. In some embodiments, therelevant stretch is at least 9, 10, 11, 12, 13, 14, 15, 16, 17 or moreresidues. In some embodiments, the relevant stretch includes contiguousresidues along a complete sequence. In some embodiments, the relevantstretch includes discontinuous residues along a complete sequence. Insome embodiments, the relevant stretch is at least 10, 15, 20, 25, 30,35, 40, 45, 50, or more residues.

The phrase “substantial identity” as used herein refers to a comparisonbetween amino acid or nucleic acid sequences. As will be appreciated bythose of ordinary skill in the art, two sequences are generallyconsidered to be “substantially identical” if they contain identicalresidues in corresponding positions. As is well known in this art, aminoacid or nucleic acid sequences may be compared using any of a variety ofalgorithms, including those available in commercial computer programssuch as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, andPSI-BLAST for amino acid sequences. Exemplary such programs aredescribed in Altschul, et al., Basic local alignment search tool, J.Mol. Biol., 215(3): 403-410, 1990; Altschul, et al., Methods inEnzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997;Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis ofGenes and Proteins, Wiley, 1998; and Misener, et al., (eds.),Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol.132), Humana Press, 1999. In addition to identifying identicalsequences, the programs mentioned above typically provide an indicationof the degree of identity. In some embodiments, two sequences areconsidered to be substantially identical if at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore of their corresponding residues are identical over a relevantstretch of residues. In some embodiments, the relevant stretch is acomplete sequence. In some embodiments, the relevant stretch is at least10, 15, 20, 25, 30, 35, 40, 45, 50, or more residues.

The phrase “targeting vector” or “targeting construct” as used hereinrefers to a polynucleotide molecule that comprises a targeting region. Atargeting region comprises a sequence that is identical or substantiallyidentical to a sequence in a target cell, tissue or animal and providesfor integration of the targeting construct into a position within thegenome of the cell, tissue or animal via homologous recombination.Targeting regions that target using site-specific recombinaserecognition sites (e.g., loxP or Frt sites) are also included. In someembodiments, a targeting construct of the present invention furthercomprises a nucleic acid sequence or gene of particular interest, aselectable marker, control and or regulatory sequences, and othernucleic acid sequences that allow for recombination mediated throughexogenous addition of proteins that aid in or facilitate recombinationinvolving such sequences. In some embodiments, a targeting construct ofthe present invention further comprises a gene of interest in whole orin part, wherein the gene of interest is a heterologous gene thatencodes a protein in whole or in part that has a similar function as aprotein encoded by an endogenous sequence.

The term “variant” as used herein refers to an entity that showssignificant structural identity with a reference entity but differsstructurally from the reference entity in the presence or level of oneor more chemical moieties as compared with the reference entity. In manyembodiments, a variant also differs functionally from its referenceentity. In general, whether a particular entity is properly consideredto be a “variant” of a reference entity is based on its degree ofstructural identity with the reference entity. As will be appreciated bythose skilled in the art, any biological or chemical reference entityhas certain characteristic structural elements. A variant, bydefinition, is a distinct chemical entity that shares one or more suchcharacteristic structural elements. To give but a few examples, a smallmolecule may have a characteristic core structural element (e.g., amacrocycle core) and/or one or more characteristic pendent moieties sothat a variant of the small molecule is one that shares the corestructural element and the characteristic pendent moieties but differsin other pendent moieties and/or in types of bonds present (single vsdouble, E vs Z, etc.) within the core, a polypeptide may have acharacteristic sequence element comprised of a plurality of amino acidshaving designated positions relative to one another in linear orthree-dimensional space and/or contributing to a particular biologicalfunction, a nucleic acid may have a characteristic sequence elementcomprised of a plurality of nucleotide residues having designatedpositions relative to on another in linear or three-dimensional space.For example, a variant polypeptide may differ from a referencepolypeptide as a result of one or more differences in amino acidsequence and/or one or more differences in chemical moieties (e.g.,carbohydrates, lipids, etc.) covalently attached to the polypeptidebackbone. In some embodiments, a variant polypeptide shows an overallsequence identity with a reference polypeptide that is at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.Alternatively or additionally, in some embodiments, a variantpolypeptide does not share at least one characteristic sequence elementwith a reference polypeptide. In some embodiments, the referencepolypeptide has one or more biological activities. In some embodiments,a variant polypeptide shares one or more of the biological activities ofthe reference polypeptide. In some embodiments, a variant polypeptidelacks one or more of the biological activities of the referencepolypeptide. In some embodiments, a variant polypeptide shows a reducedlevel of one or more biological activities as compared with thereference polypeptide. In many embodiments, a polypeptide of interest isconsidered to be a “variant” of a parent or reference polypeptide if thepolypeptide of interest has an amino acid sequence that is identical tothat of the parent but for a small number of sequence alterations atparticular positions. Typically, fewer than 20%, 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% of the residues in the variant are substituted ascompared with the parent. In some embodiments, a variant has 10, 9, 8,7, 6, 5, 4, 3, 2, or 1 substituted residue as compared with a parent.Often, a variant has a very small number (e.g., fewer than 5, 4, 3, 2,or 1) number of substituted functional residues (i.e., residues thatparticipate in a particular biological activity). Furthermore, a varianttypically has not more than 5, 4, 3, 2, or 1 additions or deletions, andoften has no additions or deletions, as compared with the parent.Moreover, any additions or deletions are typically fewer than about 25,about 20, about 19, about 18, about 17, about 16, about 15, about 14,about 13, about 10, about 9, about 8, about 7, about 6, and commonly arefewer than about 5, about 4, about 3, or about 2 residues. In someembodiments, the parent or reference polypeptide is one found in nature.As will be understood by those of ordinary skill in the art, a pluralityof variants of a particular polypeptide of interest may commonly befound in nature, particularly when the polypeptide of interest is aninfectious agent polypeptide.

The term “vector” as used herein refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it is associated.In some embodiment, vectors are capable of extra-chromosomal replicationand/or expression of nucleic acids to which they are linked in a hostcell such as a eukaryotic and/or prokaryotic cell. Vectors capable ofdirecting the expression of operatively linked genes are referred toherein as “expression vectors.”

The term “wild-type” as used herein has its art-understood meaning thatrefers to an entity having a structure and/or activity as found innature in a “normal” (as contrasted with mutant, diseased, altered,etc.) state or context. Those of ordinary skill in the art willappreciate that wild type genes and polypeptides often exist in multipledifferent forms (e.g., alleles).

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise.

DETAILED DESCRIPTION

The present invention provides, among other things, improved and/orengineered non-human animals having humanized genetic material encodingan FcγRT receptor for experimentation on human or human-like immuneeffector responses.

Fc receptors

The receptors for the Fc (i.e., constant) regions of immunoglobulins(FcRs) play an important role in the regulation of the immune response.FcRs are present on accessory cells of a host immune system tofacilitate disposal of foreign antigens bound by an antibody. FcRs alsoplay important roles in balancing both activating and inhibitoryresponses of the accessory cells of the immune system. FcRs are involvedin phagocytosis by macrophages, degranulation of mast cells, uptake ofantibody-antigen complexes and modulation of the immune response, aswell as other immune system processes.

In mice and humans, distinct FcRs are differentially expressed on thesurface of different accessory cells that are each specific for theimmunoglobulin isotypes present in the expressed antibody repertoire.For example, immunoglobulin G (IgG) antibodies mediate effectorfunctions through IgG receptors (FcγRs). FcγRs have been classified intofour groups: high affinity activating FcγRT (CD64), low affinityinhibitory FcγRIIb (CD32b), low affinity activating FcgRIIa/c (CD32a/c)and low affinity activating FcγRIII (CD 16). Although each group ispresent in both mice and humans, the number of isoforms and subsets ofimmune cells on which they are present are different. For example,FcγRIIA and FcγRIIIB are expressed on accessory cells in humans but arereportedly absent from mice. Further, affinities of the different IgGisotypes (e.g., IgG 1) for each FcγR is different between mice andhumans.

High Affinity Human FcγRI

The human high affinity FcγRT (CD64) is an integral membraneglycoprotein that binds monomeric IgG-type antibodies with high affinity(typically with Ka approximately 10⁻⁸ to 10⁻⁹M). After binding IgG, CD64interacts with an accessory chain known as the common y chain (γ chain),which possesses an immunoreceptor tyrosine-based activation motif (ITAM)that triggers cellular activation. In humans, CD64 has been reported tobe constitutively expressed on macrophages and monocytes, with inducibleexpression on polymorphonuclear leukocytes by cytokines such as IFNγ andG-CSF.

FcγRI Sequences

Exemplary sequences for human, mouse, and hybridized FcγRI are set forthin Table 3. For cDNA sequences, consecutive exons are separated byalternating underlined text. For protein sequences, extracellularsequences are underlined. The referenced sequences are exemplary; thoseskilled in the art are able to determine and compare sequence elementsor degrees of identity in order to discriminate additional mouse andhuman sequences.

TABLE 3 Mouse ACATTACATG ATTCTTACCA GCTTTGGAGA TGACATGTGG CTTCTAACAAFcγRI CTCTGCTACT TTGGGTTCCA GTCGGTGGGG AAGTGGTTAA TGCCACCAAG cDNAGCTGTGATCA CCTTGCAGCC TCCATGGGTC AGTATTTTCC AGAAGGAAAA NM_010186TGTCACTTTA TGGTGTGAGG GGCCTCACCT GCCTGGAGAC AGTTCCACACAATGGTTTAT CAACGGAACA GCCGTTCAGA TCTCCACGCC TAGTTATAGCATCCCAGAGG CCAGTTTTCA GGACAGTGGC GAATACAGGT GTCAGATAGGTTCCTCAATG CCAAGTGACC CTGTGCAGTT GCAAATCCAC AATGATTGGCTGCTACTCCA GGCCTCCCGC AGAGTCCTCA CAGAAGGAGA ACCCCTGGCCTTGAGGTGTC ACGGATGGAA GAATAAACTG GTGTACAATG TGGTTTTCTATAGAAATGGA AAATCCTTTC AGTTTTCTTC AGATTCGGAG GTCGCCATTCTGAAAACCAA CCTGAGTCAC AGCGGCATCT ACCACTGCTC AGGCACGGGAAGACACCGCT ACACATCTGC AGGAGTGTCC ATCACGGTGA AAGAGCTGTTTACCACGCCA GTGCTGAGAG CATCCGTGTC ATCTCCCTTC CCGGAGGGGAGTCTGGTCAC CCTGAACTGT GAGACGAATT TGCTCCTGCA GAGACCCGGCTTACAGCTTC ACTTCTCCTT CTACGTGGGC AGCAAGATCC TGGAGTACAGGAACACATCC TCAGAGTACC ATATAGCAAG GGCGGAAAGA GAAGATGCTGGATTCTACTG GTGTGAGGTA GCCACGGAGG ACAGCAGTGT CCTTAAGCGCAGCCCTGAGT TGGAGCTCCA AGTGCTTGGT CCCCAGTCAT CAGCTCCTGTCTGGTTTCAC ATCCTGTTTT ATCTGTCAGT GGGAATAATG TTTTCGTTGAACACGGTTCT CTATGTGAAA ATACACAGGC TGCAGAGAGA GAAGAAATACAACTTAGAAG TCCCTTTGGT TTCTGAGCAG GGAAAGAAAG CAAATTCCTTTCAGCAAGTT AGAAGCGATG GCGTGTATGA AGAAGTAACA GCCACTGCGAGCCAGACCAC ACCAAAAGAA GCGCCCGATG GACCTCGAAG CTCAGTGGGTGACTGTGGAC CCGAGCAGCC TGAACCCCTT CCTCCCAGTG ACAGTACTGGGGCACAAACT TCCCAAAGTT GACCCTGAAA CTGTGGGACC ATGGCATGCAACTCTTAAAT AAAGCAAATA TACAGACTGG ATCCGGCTGA GACAAGCTGGGTAATCAGAC ATTTGAAAGG AGACCTATAC CAAAGGGATC TTGCAACACATGGAGTCAGG TCACAGCGGG GGTTGTCGAA TGTTTGACCT TATGGAGCAGGGAAACAGGA AGTGAATCCC ACAGGACTCC CCCCCCCCGC CCATCCCCCTCCAGGCCGCC CCGGACAGGA CCCAGCTCTG GAAGACTCCA GTCTGAGACTTGCGGAACCA GAGCAGGGGT GAGATTCCTG CCCAGAAGGG ACAGCTGTGCCATCCCCTCA CAGGGTGGAT GGGTTCAGGG AAAGGCCTCC CCAGGGACGGCCTGCGTGTC AGGGGAGCAG ACGCTGATAC AGACAGCTCC ATAGCCTGGGCTAAAGCTGG CTAAGACCCG GTGGTCATCC TGAGAGCATC GGAATTTGTGCTCTCCTTCC TACCGTCTCT CTTCATGCAC CCTCCCCAGA TTTGCTGCCCACGACCCTCA AAGGACATAG TGGCGGCAGC TAAAGAGTGA AGTGTCAGCAGTAATCCATC CATCTAACCT CCCTCAGGTC CAGATACCCC CACCCCCAAACTCCCACACT CTAGGGGCCT TTTCAGGCAG CCTGCATGTG GTGTCTTAGCAGAGCTATGG TACAAAGGCT TTTAGCTCTA TCATTATCTG ACAAGCAGACAGCACCCTCA GGTGCTCTCA TTGGGTGGTG AGAGCTTTCT CCAGCCTGTACCACCTGTAA GCTGGAGTGT GGGGCGGGAA CACTGGCCCA AAGCGTCCCTATTGGAAGGC ACGGCTTACA TGGGTGTCAC AAATGCCCTT AGACCACGCAGGAAGACCGA ATTCTAGAAA CAAGGAGTAG ATCATGTCTC CACTTACTGTCACTCCTAAG GATCCCCTGA AGGTCTTGGA GCTTCACATC CCTGGAACTCTAGGGTCTGC CGTGCTAGAG GTCCCAGTCT GCAGAGTGGG TGTGGCATAGCCTGAGCCTC CCTGGATGTG AACATTAGCA AGGTATATTG GGACCTTTATAACCAGGGAC CAATAGGCAT GAGAGGGACC GGGATAATGG ACCACAGTCACAGGAGGAGA TACACTCTGT TGTACAATGC ATGCAGAAAC TGTCAAAAACAGTGTGGGAG CTGGAGAGAT GATCAGGGGT TAAGAACACT TCCTGCTCTTCCAGAGGACC TGAGTTCACT TTTTGTAACT GCTTGTAAGT CCAGATGTCGTCTTCTGATC TCTTTCAAGC ACCCACATGT GCAGGGCATG CAGACACAGACATATGAACA AGAACAATTA AAAAATAAAT TATAACTGC (SEQ ID NO: 1) MouseMILTSFGDDMWLLTTLLLWVPVGGEVVNATKAVITLQPPWVSIFQKENVTLWCE FcγRIGPHLPGDSSTQWFINGTAVQISTPSYSIPEASFQDSGEYRCQIGSSMPSDPVQL ProteinQIHNDWLLLQASRRVLTEGEPLALRCHGWKNKLVYNVVFYRNGKSFQFSSDSEV NP_034316.AILKTNLSHSGIYHCSGTGRHRYTSAGVSITVKELFTTPVLRASVSSPFPEGSL 1VTLNCETNLLLQRPGLQLHFSFYVGSKILEYRNTSSEYHIARAEREDAGFYWCEVATEDSSVLKRSPELELQVLGPQSSAPVWFHILFYLSVGIMFSLNTVLYVKIHRLQREKKYNLEVPLVSEQGKKANSFQQVRSDGVYEEVTATASQTTPKEAPDGPRSSVGDCGPEQPEPLPPSDSTGAQTSQS (SEQ ID NO: 2) HumanAATATCTTGC ATGTTACAGA TTTCACTGCT CCCACCAGCT TGGAGACAAC FcγRIATGTGGTTCT TGACAACTCT GCTCCTTTGG GTTCCAGTTG ATGGGCAAGT cDNAGGACACCACA AAGGCAGTGA TCACTTTGCA GCCTCCATGG GTCAGCGTGT NC_000001.TCCAAGAGGA AACCGTAACC TTGCACTGTG AGGTGCTCCA TCTGCCTGGG 11AGCAGCTCTA CACAGTGGTT TCTCAATGGC ACAGCCACTC AGACCTCGACCCCCAGCTAC AGAATCACCT CTGCCAGTGT CAATGACAGT GGTGAATACAGGTGCCAGAG AGGTCTCTCA GGGCGAAGTG ACCCCATACA GCTGGAAATCCACAGAGGCT GGCTACTACT GCAGGTCTCC AGCAGAGTCT TCACGGAAGGAGAACCTCTG GCCTTGAGGT GTCATGCGTG GAAGGATAAG CTGGTGTACAATGTGCTTTA CTATCGAAAT GGCAAAGCCT TTAAGTTTTT CCACTGGAATTCTAACCTCA CCATTCTGAA AACCAACATA AGTCACAATG GCACCTACCATTGCTCAGGC ATGGGAAAGC ATCGCTACAC ATCAGCAGGA ATATCTGTCACTGTGAAAGA GCTATTTCCA GCTCCAGTGC TGAATGCATC TGTGACATCCCCACTCCTGG AGGGGAATCT GGTCACCCTG AGCTGTGAAA CAAAGTTGCTCTTGCAGAGG CCTGGTTTGC AGCTTTACTT CTCCTTCTAC ATGGGCAGCAAGACCCTGCG AGGCAGGAAC ACATCCTCTG AATACCAAAT ACTAACTGCTAGAAGAGAAG ACTCTGGGTT ATACTGGTGC GAGGCTGCCA CAGAGGATGGAAATGTCCTT AAGCGCAGCC CTGAGTTGGA GCTTCAAGTG CTTGGCCTCCAGTTACCAAC TCCTGTCTGG TTTCATGTCC TTTTCTATCT GGCAGTGGGAATAATGTTTT TAGTGAACAC TGTTCTCTGG GTGACAATAC GTAAAGAACTGAAAAGAAAG AAAAAGTGGG ATTTAGAAAT CTCTTTGGAT TCTGGTCATGAGAAGAAGGT AATTTCCAGC CTTCAAGAAG ACAGACATTT AGAAGAAGAGCTGAAATGTC AGGAACAAAA AGAAGAACAG CTGCAGGAAG GGGTGCACCGGAAGGAGCCC CAGGGGGCCA CGTAGCAGCG GCTCAGTGGG TGGCCATCGATCTGGACCGT CCCCTGCCCA CTTGCTCCCC GTGAGCACTG CGTACAAACATCCAAAAGTT CAACAACACC AGAACTGTGT GTCTCATGGT ATGTAACTCTTAAAGCAAAT AAATGAACTG ACTTCAACTG GGATACATTT GGAAATGTGGTCATCAAAGA TGACTTGAAA TGAGGCCTAC TCTAAAGAAT TCTTGAAAAACTTACAAGTC AAGCCTAGCC TGATAATCCT ATTACATAGT TTGAAAAATAGTATTTTATT TCTCAGAACA AGGTAAAAAG GTGAGTGGGT GCATATGTACAGAAGATTAA GACAGAGAAA CAGACAGAAA GAGACACACA CACAGCCAGGAGTGGGTAGA TTTCAGGGAG ACAAGAGGGA ATAGTATAGA CAATAAGGAAGGAAATAGTA CTTACAAATG ACTCCTAAGG GACTGTGAGA CTGAGAGGGCTCACGCCTCT GTGTTCAGGA TACTTAGTTC ATGGCTTTTC TCTTTGACTTTACTAAAAGA GAATGTCTCC ATACGCGTTC TAGGCATACA AGGGGGTAACTCATGATGAG AAATGGATGT GTTATTCTTG CCCTCTCTTT TGAGGCTCTCTCATAACCCC TCTATTTCTA GAGACAACAA AAATGCTGCC AGTCCTAGGCCCCTGCCCTG TAGGAAGGCA GAATGTAACT GTTCTGTTTG TTTAACGATTAAGTCCAAAT CTCCAAGTGC GGCACTGCAA AGAGACGCTT CAAGTGGGGAGAAGCGGCGA TACCATAGAG TCCAGATCTT GCCTCCAGAG ATTTGCTTTACCTTCCTGAT TTTCTGGTTA CTAATTAGCT TCAGGATACG CTGCTCTCATACTTGGGCTG TAGTTTGGAG ACAAAATATT TTCCTGCCAC TGTGTAACATAGCTGAGGTA AAAACTGAAC TATGTAAATG ACTCTACTAA AAGTTTAGGGAAAAAAAACA GGAGGAGTAT GACACAAAAA AAAAAAAAAA AAAAAAAAAAAAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAAAAAAAAAAAA AAAAAAAA (SEQ ID NO: 3) HumanMWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQ FcγRIWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVS ProteinSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGT AAI52384YHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWFHVLFYLAVGIMELVNTVLWVTIRKELKRKKKWDLEISLDSGHEKKVISSLQEDRHLEEELKCQEQKEEQLQEGVHRKEPQGAT (SEQ ID NO: 4) ExemplaryMWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQ HumanizedWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVS FcγRISRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGT ProteinYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWGPQSSAPVWFHILFYLSVGIMFSLNTVLYVKIHRLQREKKYNLEVPLVSEQGKKANSFQQVRSDGVYEEVTATASQTTPKEAPDGPRSSVGDCGPEQPEPLPPSDSTGAQTSQS (SEQ ID NO: 5)

Humanized FcγRI Non-Human Animals

Non-human animals are provided that express humanized FcγRI receptorproteins on the surface of immune cells (e.g., myeloid cells) of thenon-human animals resulting from a genetic modification of an endogenouslocus of the non-human animal that encodes an FcγRI protein. Suitableexamples described herein include rodents, in particular, mice.

A humanized endogenous FcγRI gene, in some embodiments, comprisesgenetic material from a heterologous species (e.g., humans), wherein thehumanized endogenous FcγRI gene encodes a FcγRI protein that comprisesthe encoded portion of the genetic material from the heterologousspecies. In some embodiments, a humanized endogenous FcγRI gene of thepresent invention comprises genomic DNA of a heterologous species thatcorresponds to the extracellular portion of a FcγRI protein that isexpressed on the plasma membrane of a cell. Non-human animals, embryos,cells and targeting constructs for making non-human animals, non-humanembryos, and cells containing said humanized endogenous FcγRI gene arealso provided.

In some embodiments, the endogenous FcγRI locus is deleted. In someembodiments, the endogenous FcγRI locus is altered, wherein a portion ofthe endogenous FcγRI locus is replaced with a heterologous sequence(e.g., a human FcγRI sequence in whole or in part). In some embodiments,all or substantially all of the endogenous FcγRI locus is replaced witha heterologous locus (e.g., a human FcγRI locus). In some embodiments, aportion of a heterologous FcγRI locus is inserted into an endogenousnon-human FcγRI locus. In some embodiments, the heterologous locus is ahuman locus.

A non-human animal of the present invention contains a human FcγRI genein whole or in part at an endogenous non-human FcγRI locus. Thus, suchnon-human animals can be described as having a heterologous FcγRI gene.The replaced, inserted or modified endogenous FcγRI locus can bedetected using a variety of methods including, for example, PCR, Westernblot, Southern blot, restriction fragment length polymorphism (RFLP), ora gain or loss of allele assay.

In various embodiments, a humanized FcγRI gene according to the presentinvention includes a FcγRI gene that has a third, fourth and fifth exoneach having a sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to a third, fourth, and fifth exon that appear in a humanFcγRI gene of SEQ ID NO: 3.

In various embodiments, a humanized FcγRI gene according to the presentinvention includes a FcγRI gene that has a nucleotide coding sequence(e.g., a cDNA sequence) at least 50% (e.g., 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to nucleotides that appear in SEQ ID NO: 5.

In various embodiments, a humanized FcγRI protein produced by anon-human animal of the present invention has an extracellular portionhaving a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to an extracellular portion of a human FcγRI protein thatappears in Table 3.

In various embodiments, a humanized FcγRI protein produced by anon-human animal of the present invention has an extracellular portionhaving a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to amino acid residues 18-288 that appear in a human FcγRIprotein of SEQ ID NO: 4.

In various embodiments, a humanized FcγRI protein produced by anon-human animal of the present invention has an amino acid sequencethat is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to anamino acid sequence of a humanized FcγRI protein as exemplified in SEQID NO: 5.

Compositions and methods for making non-human animals that expresses ahumanized FcγRI protein, including specific polymorphic forms or allelicvariants (e.g., single amino acid differences), are provided, includingcompositions and methods for making non-human animals that expressessuch proteins from a human promoter and a human regulatory sequence. Insome embodiments, compositions and methods for making non-human animalsthat expresses such proteins from an endogenous promoter and anendogenous regulatory sequence are also provided. The methods includeinserting the genetic material encoding a human FcγRI protein in wholeor in part at a precise location in the genome of a non-human animalthat corresponds to an endogenous FcγRI gene thereby creating ahumanized FcγRI gene that expresses a FcγRI protein that is human inwhole or in part. In some embodiments, the methods include insertinggenomic DNA corresponding to exons 3-5 a humanized gene that encodes aFcγRI protein that contains a human portion containing amino acidsencoded by the inserted exons.

A humanized endogenous FcγRI gene approach employs a relatively minimalmodification of the endogenous gene and results in natural FcγRImediated effector responses in the non-human animal, in variousembodiments, because the genomic sequence of the FcγRI sequences aremodified in a single fragment and therefore retain normal functionalityby including necessary regulatory sequences. Thus, in such embodiments,the FcγRI gene modification does not affect other surrounding genes orother endogenous FcγRI genes. Further, in various embodiments, themodification does not affect the assembly of a functional receptor onthe plasma and maintains normal effector functions via binding andsubsequent signal transduction through the cytoplasmic portion of thereceptor which is minimally or unaffected by the modification.

A schematic illustration (not to scale) of an endogenous murine FcγRIgene and a humanized endogenous FcγRI gene is provided in FIG. 5. Asillustrated, genomic DNA containing exons 3-5 of a human FcγRI gene isinserted into an endogenous murine FcγRI gene locus by a targetingconstruct. This genomic DNA includes comprises the portion of the genethat encodes one or more extracellular domain regions (e.g., amino acidresides 28-362) of a human FcγRT protein that participate in Fc binding.

A non-human animal (e.g., a mouse) having a humanized endogenous FcγRIgene can be made by any method known in the art. For example, atargeting vector can be made that introduces a human FcγRT gene in wholeor in part with a selectable marker gene. FIG. 5 illustrates a mousegenome comprising an insertion of exons 1-5 of a human FcγRT. Asillustrated, the targeting construct contains a 5′ homology armcontaining sequence upstream of exon 1 of an endogenous murine FcγRTgene, followed by a genomic DNA fragment containing exons 1-5 of a humanFcγRT gene, a drug selection cassette (e.g., a neomycin resistance geneflanked on both sides by loxP sequences), and a 3′ homology armcontaining sequence downstream of exons 6 of an endogenous murine FcγRTgene. Upon homologous recombination, exons 1-5 and a portion of exon 6of an endogenous murine FcγRT gene is replaced by the sequence containedin the targeting vector. A humanized endogenous FcγRT gene is createdresulting in a cell or non-human animal that expresses a humanized FcγRTprotein that contains amino acids encoded by exons 1-5 of a human FcγRTgene. The drug selection cassette may optionally be removed by thesubsequent addition of a recombinase (e.g., by Cre treatment).

In addition to mice having humanized FcγRI genes as described herein,also provided herein are other genetically modified non-human animalsthat comprise humanized FcγRI genes. In some embodiments, such non-humananimals comprise a humanized FcγRI gene operably linked to an endogenousFcγRI promoter. In some embodiments, such non-human animals express ahumanized FcγRI protein from an endogenous locus, wherein the humanizedFcγRI protein comprises amino acid residues 16-290 of a human FcγRIprotein.

Such non-human animals may be selected from the group consisting of amouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep,goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesusmonkey). For the non-human animals where suitable genetically modifiableES cells are not readily available, other methods are employed to make anon-human animal comprising genetic modifications as described herein.Such methods include, e.g., modifying a non-ES cell genome (e.g., afibroblast or an induced pluripotent cell) and employing nucleartransfer to transfer the modified genome to a suitable cell, e.g., anoocyte, and gestating the modified cell (e.g., the modified oocyte) in anon-human animal under suitable conditions to form an embryo.

In some embodiments, a non-human animal of the present invention is amammal. In some embodiments, a non-human animal of the present inventionis a small mammal, e.g., of the superfamily Dipodoidea or Muroidea. Insome embodiments, a genetically modified animal of the present inventionis a rodent. In some embodiments, a rodent of the present invention isselected from a mouse, a rat, and a hamster. In some embodiments, arodent of the present invention is selected from the superfamilyMuroidea. In some embodiments, a genetically modified animal of thepresent invention is from a family selected from Calomyscidae (e.g.,mouse-like hamsters), Cricetidae (e.g., hamster, New World rats andmice, voles), Muridae (true mice and rats, gerbils, spiny mice, crestedrats), Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasyrats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae(e.g., mole rates, bamboo rats, and zokors). In some certainembodiments, a genetically modified rodent of the present invention isselected from a true mouse or rat (family Muridae), a gerbil, a spinymouse, and a crested rat. In some certain embodiments, a geneticallymodified mouse of the present invention is from a member of the familyMuridae. In some embodiment, an non-human animal of the presentinvention is a rodent. In some certain embodiments, a rodent of thepresent invention is selected from a mouse and a rat. In someembodiments, a non-human animal of the present invention is a mouse.

In some embodiments, a non-human animal of the present invention is arodent that is a mouse of a C57BL strain selected from C57BL/A,C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ,C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In somecertain embodiments, a mouse of the present invention is a 129 strainselected from the group consisting of a strain that is 129P1, 129P2,129P3,129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm), 129S2, 129S4, 129S5,129S9/SvEvH, 129/SvJae, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2(see, e.g., Festing et al., 1999, Mammalian Genome 10:836; Auerbach etal., 2000, Biotechniques 29(5):1024-1028, 1030, 1032). In some certainembodiments, a genetically modified mouse of the present invention is amix of an aforementioned 129 strain and an aforementioned C57BL/6strain. In some certain embodiments, a mouse of the present invention isa mix of aforementioned 129 strains, or a mix of aforementioned BL/6strains. In some certain embodiments, a 129 strain of the mix asdescribed herein is a 129S6 (129/SvEvTac) strain. In some embodiments, amouse of the present invention is a BALB strain, e.g., BALB/c strain. Insome embodiments, a mouse of the present invention is a mix of a BALBstrain and another aforementioned strain.

In some embodiments, a non-human animal of the present invention is arat. In some certain embodiments, a rat of the present invention isselected from a Wistar rat, an LEA strain, a Sprague Dawley strain, aFischer strain, F344, F6, and Dark Agouti. In some certain embodiments,a rat strain as described herein is a mix of two or more strainsselected from the group consisting of Wistar, LEA, Sprague Dawley,Fischer, F344, F6, and Dark Agouti.

Non-Human Animals Having Humanized FcγRI Genes

FcγR mutant and transgenic non-human animals (e.g., mice) have beenreported, for example, by van de Winkel et al. in U.S. Pat. No.6,111,166.

Such animals have been employed in assays to assess the molecularaspects of FcγRI expression, function and regulation. However, they arenot without limitations and disadvantages. For example, the mousedisclosed in the '166 patent contains human FcγRI randomly inserted intoits genome, which (1) may disrupt the expression and or function ofother genes unintentionally, whether detected or not, and (2) results inexpression of both fully human and fully mouse FcγRI which complicatesor confounds particular study of a single type of FcγR. Moreover,intracellular signaling region of FcγRIα chain may be perturbed,disrupted, or otherwise not in accordance with normal FcγRI in the mousedisclosed in the '116 patent because the intracellular region of theFcγRIα chain that participates in signal transduction is human ratherthan mouse.

The present invention provides a means to overcome these and otherdisadvantages. The present invention provides, among other things, ahumanized FcγRI transgene inserted at an endogenous mouse locus toreplace mouse FcγRI with a human or hybrid FcγRI gene. In someembodiments, a hybrid FcγRI gene is inserted at the endogenous mouselocus, wherein the extracellular domain comprises a human sequence andthe intracellular domain comprises a mouse sequence. In someembodiments, insertion of a hybrid FcγRI gene at an endogenous locus ofmouse FcγRI gene provides expression of FcγRI protein on immune cellsthat more closely resembles the distribution of human FcγRI protein onhuman immune cells as compared to the distribution of human FcγRIprotein expression on immune cells of a mouse that additionallyexpresses an endogenous mouse FcγRI protein. In some embodiments,insertion of a hybrid FcγRI gene at an endogenous locus of mouse FcγRIgene provides expression of FcγRI protein on immune cells that isinduced and regulated by appropriate signals and stimuli.

In some embodiments, FcγRIα chain mediated presentation of MHC class IIantigens is functionally maintained in mice having a hybrid FcγRIprotein with a humanized extracellular region a mouse FcγRIα chainintracellular region. In some embodiments, intracellular processing ofinternalized FcγRT is preserved in a mouse having a hybrid FcγRT proteinwith a mouse intracellular region as compared to that in a mouse havinga fully human FcγRT protein or an FcγRI protein with a non-murineintracellular region.

Non-human animals of the present invention provide an improved in vivosystem and source of biological materials (e.g., cells) expressing humanFcγRT that are useful for a variety of assays. In various embodiments,non-human animals of the present invention are used to developtherapeutics that target FcγRI and/or modulate FcγRI signaling andimmune effector responses. In various embodiments, mice of the presentinvention are used to screen and develop candidate therapeutics (e.g.,antibodies) to which FcγRI binds. In various embodiments, non-humananimals of the present invention are used to determine the immuneeffector response associated with a particular therapeutic antibody.

Genetically modified non-human animals that do not express endogenoushigh affinity mouse FcγR genes are useful, e.g., to elucidate thevarious functions of the individual high affinity FcγR genes in theimmune response, to measure the efficacy of a human therapeutic antibodyvia cell-mediated immunity (e.g., ADCC), to determine a role of FcγR inimmune diseases or disorders, to serve as models of immune diseases ordisorders, to generate antibodies against one or more FcγR proteins, andto serve as breeding mates to generate other genetically modified miceof interest.

In one embodiment, a mouse according to the invention can be used todetermine a cytotoxic effect lost (in comparison to a wild type mouse)by a mouse that does not express high affinity FcγR genes byadministering an agent to such a mouse, where the agent is known totrigger an FcγR dependent cytotoxic effect in wild type mice. In oneembodiment, a mouse of the present invention is implanted with tumorcells and, after a subsequent period of time, injected with an antibodyspecific for an antigen expressed on the surface of the tumor cells. Theisotype of the antibody is known prior to injection and the animals areanalyzed for impairment of FcγR-dependent ADCC by comparison to ADCCobserved in wild type animals.

In one aspect, mice deficient in endogenous high affinity receptorscould be combined (e.g., by breeding) with other immune deficient miceto develop in vivo models of autoimmune disease. For example, SevereCombined Immunodeficiency (SCID) mice are routinely used in the art asmodel organisms for studying the inner system. Scm mice have an impairedability to make Tor B lymphocytes, or activate some components of thecomplement system, and cannot efficiently fight infections, rejecttumors, and reject transplants. High affinity FcγR a-subunitgene-deficient mice of the present invention may be bred to SCID mice toascertain cell depletion in a host animal in response to administrationof an antibody therapeutic (e.g., an anti-tumor antibody), which woulddetermine the roles of ADCC and complement dependent cytotoxicity (CDC)in tumor cell depletion in vivo.

In some embodiments, genetically modified non-human animals comprising areplacement of the endogenous high affinity FcγR genes withhigh-affinity human FcγR genes are provided. Such animals are useful forstudying the pharmacokinetics of fully human antibodies andFcγR-mediated ADCC. In addition, human FcγR genes have been shown toexhibit polymorphisms or allelic variants associated with disease. Thus,genetically modified non-human animals that comprise a replacement ofthe endogenous high affinity FcγR genes with specific allelic orpolymorphic forms of human FcγR genes can be used to study humanautoimmune diseases, and traits associated with the polymorphisms, inthe animal. In some embodiments, the allelic forms of human FcγR genesare associated with enhanced efficacy for human IgG.

In some embodiments, the effect of a human high affinity FcγRpolymorphism on the efficacy of a human antibody therapeutic isdetermined. In some embodiments, an anti-tumor antibody is administeredto a first humanized mouse comprising a first polymorphism of a humanFcγR and also to a second humanized mouse comprising a secondpolymorphism of a human FcγR, wherein the first and the second mice eachcomprise a human tumor cell; and the anti-tumor activity of theanti-tumor antibody is assessed in the first mouse and in the secondmouse.

In some embodiments, a treatment option is selected by a physician withrespect to treating a human having the first or the second polymorphismand having a tumor corresponding to the human tumor cell based on theassessment of efficacy of the anti-tumor antibody in the first mouse andin the second mouse.

The endogenous FcγRI α-chain replacement approach employs a relativelyminimal disruption of natural FcγR-mediated signal transduction in theanimal. In various embodiments, genomic sequence of the FcγR α-chainsare replaced in a single fragment and therefore retain normalfunctionality by including necessary regulatory sequences. Thus, in suchembodiments, the FcγR α-chain modification does not impair otherendogenous FcRs dependent upon functional FcRγ-chain molecules. Further,in various embodiments, the modification does not affect the assembly offunctional receptor complex involving an FcγR α-chain and the endogenousFcRγ-chain, which may be important for proper expression of some FcγRα-chains on the cell surface and for certain downstream signalingresulting from an activated receptor. Because the FcR γ-chain is notdeleted, in various embodiments animals containing a replacement ofendogenous FcγR α-chain genes with human FcγR α-chain genes may processnormal effector functions from antibodies through binding of the Fcportion of IgG immunoglobulins to the human FcγR α-chains present on thesurface of accessory cells.

Non-human animals of the present invention express humanized FcγRTprotein, thus cells, cell lines, and cell cultures can be generated toserve as a source of humanized FcγRI for use in binding and functionalassays, e.g., to assay for binding or function of FcγRT to a potentialtherapeutic antibody. In various embodiments, a humanized FcγRI proteinexpressed by a non-human animal as described herein may comprise avariant amino acid sequence. Variant human FcγRI proteins havingvariations associated with ligand binding residues have been reported.In various embodiments, non-human animals of the present inventionexpress a humanized FcγRI protein variant. In various embodiments, thevariant is polymorphic at an amino acid position associated with ligandbinding. In various embodiments, non-human animals of the presentinvention are used to determine the immune effector response of atherapeutic antibody through interaction with a polymorphic variant ofhuman FcγRI.

Cells from non-human animals of the present invention can be isolatedand used on an ad hoc basis, or can be maintained in culture for manygenerations. In various embodiments, cells from a non-human animal ofthe present invention are immortalized and maintained in cultureindefinitely (e.g., in serial cultures).

Non-human animals of the present invention provide improved in vivosystem elucidating mechanisms of antibody dependent cell mediatedcytotoxicity.

EXAMPLES

The following examples are provided so as to describe to those ofordinary skill in the art how to make and use methods and compositionsof the invention, and are not intended to limit the scope of what theinventors regard as their invention. Unless indicated otherwise,temperature is indicated in Celsius, and pressure is at or nearatmospheric.

Example 1 Humanization of an Endogenous FcγRI Gene.

This example illustrates exemplary methods of humanizing an endogenousgene encoding high affinity FcγRI in a non-human mammal such as a rodent(e.g., a mouse).

Construction of Humanization FcγRI Targeting Vector (MAID6074)

A large targeting vector (LTVEC) was constructed by using the human FcGamma Receptor 1 gene (promoter region, signal plus ecto domain region)to replace the mouse counterparts sequence on murine chromosome 3.

Generation of BAC-Based Targeting Vectors (MAID6073)

Mouse BAC RP23-477p23 and human BAC CTD-2339o22 containing the gene ofFcγRI were identified using blast and BAC end sequence based on databaseof NCBI and Ensemble.

The approach to generate targeting vectors, the LTVEC contained humansequence from (5′ distal end) FcγRI gene promoter (25kb) to the 3′proximal end at the gene codon W290 (before its transmembranedomain(TM)) of the human FcγRI gene, (followed by the mousetrans-membrane domain and the rest of gene), involves the followingsteps.

First, by homologous recombination in bacteria, 5′ end of human sequencefrom human BAC(CTD-2339o22) removed and left a I-Ceul site and a 25 kbpromoter region of the FcγRI gene, 3′ end human sequence removed by theloxped pgk-Neo cassette located in the intron 5 (404bp upstream of TMdomain) of FcγRI gene, followed by AsiS1 site.

Second, by homologous recombination in bacteria, in Mouse BACRP23-477p23, mouse FcγRI gene (from its promoter(20 kb) up to thetransmembrane domain) removed by a Spec cassette (8 AA of EC3 domain ofhuman FcγRI sequence (up to the codon W290) added before mouse TM)flanked by I-Ceul and AsiS1 sites.

Third, digestion and ligation by I-Ceul and AsiS1 sites to generate theLTVEC (MAID6073) contained human sequence from the (5′ distal end) FcγRIgene promoter (25 kb) to the 3′ proximal end at the codon W290 (beforethe transmembrane domain) of the human FcγRI, followed by the mousetransmembrane domain and the rest of gene.

FIG. 32 depicts a schematic for and exemplary strategy for thehumanization of mouse FcγRI. MAID6074 is the cassette removed version ofMAID6073. The junction sequences are shown in FIG. 6.

Selection of Targeted Mouse ES Cells

The MAID 6074 LTVEC was electroporated into the mouse ES cell line F1H4.

Example 2 Generation of High Affinity FcγRI Humanized Mice

This example illustrates transformation and breeding of mice. hFcgR1ecto domain (MAID 6073) LTVEC was electroporated into parental F1H4mouse Embryonic Stem (ES)cells. Colonies surviving G418 drug selectionwere picked and screened for the homologous recombination of human FcγRIsequence into the FcγRI mouse locus. Eight clones were identified tohave the appropriate modification and being heterozygous for human FcγRIecto domain, one of which was clone 6073F-D2. All these clones containedthe neo cassette.

Clone 6073F-D2 was electroporated with 2 μg of Cre plasmid to remove theneo cassette. The colonies were picked and then screened for the absenceof the neo cassette. Of the clones determined to have the neo cassetteremoved, ES clones 6074B-A1 and 6074B-A10 were micro-injected using theVelociMouse method.

Male and female (XY female) FO VelociMice were generated from clones6074B-A1 and 6074B-A10. These FO mice were bred to each other in clonaland non-clonal pairings. Fl mice were produced and these mice were shownto be heterozygous and homozygous (and wildtype) for the human FcγRI.The Fl mice appeared normal and the ratio of Hom:Het:WT mice followedthe predicted Mendelian ratio of 1:2:1. Cohorts of males and femalesbeing wild-type and homozygous for human FcγRI were transferred forstudy.

Example 3 Characterization of High Affinity FcγRI Humanized Mice

This example illustrates the characteristic expression of high affinityFcγRI protein on the surface of cells from non-human animals engineeredto contain an humanized FcγRI gene construct as described in Example 1at an endogenous FcγRI locus.

Genotypic characterization of high affinity FcγRI humanized mice isshown in FIGS. 1-3. Transcript for mouse FcγRI is not detected in micehomozygous for humanized FcγRI.

The results of phenotypic characterization of are shown in FIGS. 4, and7-16.

Example 4 Phenotypic Characterization of High Affinity FcγRI HumanizedMice Treated with Murine Granulocyte Colony Stimulating Factor (mG-CSF)

Phenotypic analysis was performed for MAID 6074 (hFcγRI) HO mice treatedwith murine G-CSF (mG-CSF) vs. phosphate buffered saline (PBS) control.Mice were examined 48 hours after subcutaneous injection. Data is shownfor 6-7 week old MAID 6074 WT mice treated with PBS (n=2) or mG-CSF(n=2) compared to MAID 6074 HO mice treated with PBS (n=2) or mG-CSF(n=3). Results were similar for 16-17 week old MAID 6074 WT mice treatedwith PBS (n=1) or mG-CSF (n=1) compared to MAID 6074 HO mice treatedwith PBS (n=1) or mG-CSF (n=1). Baseline (PBS) and mG-CSF inducedexpression of mouse or hybridized FcγRI in monocytes, macrophages,neutrophils and dendritic cells in blood and spleen of MAID 6074 WT andMAID 6074 HO mice are shown in FIGS. 17-31.

Untreated MAID 6074 HO mice express FcγRI (CD64) mRNA in blood, althoughprotein was not detected by FACS. G-CSF (48 hrs) induced an increase inFcγRI (CD64) mRNA in blood and spleen, and protein as detected by FACS.

Example 5 Phenotypic Characterization of Mice Expressing Humanized Highand Low Affinity Fcγ Receptors

Mice were generated that expressed humanized high affinity and lowaffinity Fcγ receptors by using standard breeding techniques.Specifically, mice expressing humanized high affinity FcγRI generated asdescribed in Examples 1 and 2 were crossed with mice expressinghumanized low affinity FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and FcγRIIIbgenerated as described in Examples 1-6 and FIGS. 1-6 of U.S. Pat. App.Pub. No. 2014/0154701, which is hereby incorporated by reference. Theresulting mice were bred to homozygosity.

Phenotypic analysis was performed on the humanized high and low affinityFcγR mice following treatment with murine G-CSF (mG-CSF) or a phosphatebuffered saline (PBS) control. Mice were administered a subcutaneousinjection of PBS or mG-CSF (62 μg s.c., single dose). After 48 hours,blood and spleens from the treated mice were harvested andphenotypically characterized by FACS as described in Example 4. The cellsurface phenotype of the humanized high and low affinity FcγR mice wassimilar to the phenotype observed for the high affinity FcγR humanizedmice. Also similar to the high affinity Fcγ receptor humanized mice, themice in which both the high and low affinity Fcγ receptor were humanizedshowed increased expression of human FcγRI in blood and splenicmonocytes, macrophages and neutrophils. In summary, humanization of thelow affinity Fcγ receptors in the FcγRI humanized mice produced nosignificant phenotypic change.

Equivalents

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated by those skilled in the art thatvarious alterations, modifications, and improvements will readily occurto those skilled in the art. Such alterations, modifications, andimprovements are intended to be part of this disclosure, and areintended to be within the spirit and scope of the invention.Accordingly, the foregoing description and drawing are by way of exampleonly and the invention is described in detail by the claims that follow.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention can be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification.

Those skilled in the art will appreciate typical standards of deviationor error attributable to values obtained in assays or other processesdescribed herein.

What is claimed is:
 1. A mouse that expresses an FcγRI proteincomprising an extracellular portion of a human FcγRI α chain and anintracellular portion of a mouse FcγRI α chain.
 2. The mouse of claim 1,wherein the extracellular portion of a human FcγRI α chain comprises anEC1 domain, EC2 domain, an EC3 domain, or a combination thereof.
 3. Themouse of claim 2, wherein the EC1 domain is encoded by an exon at least90% identical to exon 3 of SEQ ID NO:
 3. 4. The mouse of claim 2,wherein the EC2 domain is encoded by an exon at least 90% identical toexon 4 of SEQ ID NO:
 3. 5. The mouse of claim 2, wherein the EC3 domainis encoded by an exon at least 90% identical to exon 5 of SEQ ID NO: 3.6. The mouse of claim 1, wherein the mouse does not detectably express afull-length mouse FcγRI α chain.
 7. The mouse of claim 1, wherein theintracellular portion of an FcγRI α chain comprises a cytoplasmic domainof a mouse FcγRI α chain in whole or in part.
 8. The mouse of claim 1,wherein the FcγRI protein further comprises a mouse FcγRI α chaintransmembrane domain in whole or in part.
 9. The mouse of claim 1,wherein the FcγRI protein is expressed on monocytes, macrophages,neutrophils or dendritic cells.
 10. The mouse of claim 9, wherein theexpression of the FcγRI protein is increased upon administration ofmurine granulocyte colony stimulating factor (mG-CSF) to the mouse. 11.A mouse comprising an FcγRI gene that comprises at least one exon of ahuman FcγRI gene encoding an extracellular portion of human FcγRIprotein operably linked to at least one exon of a mouse FcγRI geneencoding an intracellular portion of a mouse FcγRI protein.
 12. Themouse of claim 11, wherein the exon of the human FcγRT gene is selectedfrom the group consisting of exons 3, 4 and
 5. 13. The mouse of claim11, wherein the mouse does not express a functional mouse FcγRT gene.14. An embryonic stem cell whose genome comprises a FcγRT gene thatencodes an extracellular portion of a human FcγRT protein and anintracellular portion of a mouse FcγRT protein.
 15. The cell of claim14, wherein the FcγRT gene comprises exons 3, 4, and 5 of a human FcγRTgene.
 16. The cell of claim 14, wherein the FcγRT gene further comprisesone or more human 5′ untranslated regions flanking human exon
 1. 17. Thecell of claim 14, wherein the extracellular portion of the human FcγRIprotein comprises one or more of EC1, EC2, and EC3.
 18. The cell ofclaim 14, wherein the FcγRT gene comprises exon 6 of a mouse FcγRT gene.19. The cell of claim 14, wherein the intracellular portion of the mouseFcγRT protein comprises the cytoplasmic domain of a mouse FcγRI proteinin whole or in part.
 20. The cell of claim 14, wherein the FcγRT gene ispositioned at an endogenous FcγRT locus.
 21. A nucleic acid encoding anFcγRT protein comprising an extracellular portion of a human FcγRT αchain and an intracellular portion of a mouse FcγRT α chain.
 22. Thenucleic acid of claim 21, wherein the extracellular portion of a humanFcγRI α chain comprises an EC1 domain, EC2 domain, an EC3 domain, or acombination thereof.
 23. The nucleic acid of claim 22, wherein the EC1domain is encoded by a sequence at least 90% identical to exon 3 of SEQID NO:
 3. 24. The nucleic acid of claim 22, wherein the EC2 domain isencoded by a sequence at least 90% identical to exon 4 of SEQ ID NO: 3.25. The nucleic acid of claim 22, wherein the EC3 domain is encoded by asequence at least 90% identical to exon 5 of SEQ ID NO:
 3. 26. Thenucleic acid of claim 21, wherein the FcγRI protein comprises an FcγRI αchain amino acid sequence at least 90% identical to SEQ ID NO:
 5. 27.The nucleic acid of claim 21, wherein the FcγRI protein comprises anFcγRI α chain amino acid sequence of SEQ ID NO:
 5. 28. The nucleic acidof claim 21, wherein the nucleic acid is a vector.
 29. The nucleic acidof claim 28, wherein the vector is a targeting vector.
 30. A cellcomprising the nucleic acid of claim
 21. 31. The cell of claim 30,wherein the cell is part of a tissue.
 32. An FcγRI protein comprising anextracellular portion of a human FcγRI α chain and an intracellularportion of a mouse FcγRI α chain.
 33. The FcγRI protein of claim 32,wherein the extracellular portion of the human FcγRI α chain comprisesan EC1 domain, EC2 domain, an EC3 domain, or a combination thereof. 34.The FcγRI protein of claim 33, wherein the EC1 domain is encoded by asequence at least 90% identical to exon 3 of SEQ ID NO:
 3. 35. The FcγRIprotein of claim 33, wherein the EC2 domain is encoded by a sequence atleast 90% identical to exon 4 of SEQ ID NO:
 3. 36. The FcγRI protein ofclaim 33, wherein the EC3 domain is encoded by a sequence at least 90%identical to exon 5 of SEQ ID NO:
 3. 37. The FcγRI protein of claim 32,wherein the FcγRI protein comprises an FcγRI α chain amino acid sequenceat least 90% identical to SEQ ID NO:
 5. 38. The FcγRI protein of claim32, wherein the FcγRI protein comprises an FcγRI α chain amino acidsequence of SEQ ID NO:
 5. 39. A cell comprising the FcγRI protein ofclaim 32.